Endotoxin binding and neutralizing protein and uses thereof

ABSTRACT

Endotoxin binding/neutralizing proteins capable of binding endotoxin in vivo, thereby neutralizing the toxic effect or bioactivity of endotoxin which are isolated from a horseshoe crab such as Limulus polyphemus, pharmaceutical compositions and pharmaceutical uses of the proteins, a method of purifying the proteins and an assay for endotoxin based on the proteins, are disclosed.

This application is a division of application Ser. No. 08/264,244, filedJun. 22, 1994, now U.S. Pat. No. 5,599,113, which is a continuation ofapplication Ser. No. 07/883,457, filed May 15, 1992, abandoned, which isa continuation-in-part of application Ser. No. 07/701,501, filed May 16,1991, now abandoned which is a continuation-in-part of application Ser.No. 07/480,957, filed Feb. 16, 1990, now abandoned which is a divisionalof application Ser. No. 07/210,575, filed Jun. 23, 1988 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to pharmaceutical compositions anduses of an endotoxin binding/neutralizing protein which may be isolatedfrom a horseshoe crab. The invention covers, inter alia, pharmaceuticalcompositions and pharmaceutical uses of the protein, a method ofpurifying the protein, and an assay for endotoxin based on this protein.

2. Discussion of the Background

Despite aggressive management, septic shock arising from gram-negativesepsis continues to be a leading cause of death in both surgical andmedical patients. Death in such patients usually results fromcardiovascular collapse and/or multiple organ system failure. One of themain components of gram-negative bacteria thought to play an integralrole in causing septic shock is an outer wall constituent, endotoxin.

Endotoxins are high molecular weight complexes, associated with theouter membrane of gram-negative bacteria, that produce pyrogenicreactions upon intravenous administration. Endotoxin is shed from livingbacteria and is also released into the environment when bacteria die anddecompose. Since gram-negative bacteria are found in great numbers inair, water, and soil, bacterial endotoxin commonly contaminates rawmaterials and processing equipment used in the manufacturing of, forexample, pharmaceuticals.

Bacterial endotoxin is a complex consisting of lipid, carbohydrate andprotein. It is characterized by an overall negative charge, heatstability and high molecular weight. Highly purified endotoxin does notcontain protein, and is a lipopolysaccharide (LPS). Depyrogenation cangenerally be achieved by inactivating or removing endotoxin, dependingupon the physicochemical nature of the LPS. LPS consists of threedistinct chemical regions, lipid A, which is the innermost region, anintermediate core polysaccharide, and an outermost 0-specificpolysaccharide side chain which is responsible for an endotoxin'sparticular immunospecificity.

Bacterial endotoxins are known to have profound biological effects inanimals and humans, and to cause severe medical problems when present.Symptoms include induction of high fever, activation of complement, andhypotension. It is critical to avoid endotoxin contamination in anypharmaceutical product or medical device which comes into contact withbody fluids. High endotoxin levels in sera due to bacterial diseases,such as septicemia, are not easily treated. Antibiotic treatment of theinfection only kills the bacteria, leaving the endotoxin from their cellwalls free to cause fever.

The horseshoe crab Limulus polyphemus is particularly sensitive toendotoxin. The cells from their hemolymph (amebocytes) undergo a complexseries of biochemical reactions resulting in clot formation, analogousto mammalian blood coagulation. This phenomenon has been exploited inthe form of bioassays sensitive to very low endotoxin levels. Currently,a bioassay of this type is the method of choice for monitoringpharmaceutical manufacturing and is termed Limulus Amebocyte Lysate(LAL). See U.S. Pat. Nos. 4,276,050, 4,273,557, 4,221,866, 4,201,865,4,038,147, 3,944,391 and 3,915,805, each of which is incorporated hereinby reference.

It has long been observed that once endotoxin interacts with LAL thetoxin is not recoverable from the clot. See Nachum et al, Journal ofInvertebrate Pathology, 32:51-58 (1978). This observation ledinvestigators to postulate two alternative explanations. Either theendotoxin is enzymatically degraded during clot formation or it is boundby some factor causing it to lose toxicity. The present inventorsinitiated experiments to extract the endotoxin inactivating factor fromthe LAL.

Other research groups have experimented with endotoxin binding proteins,also referred to as anti-LPS factor. To the inventor's knowledge, thefollowing publications resulting from work in this area are the mostrelevant to this invention:

Tanaka et al, Biochem. Biophys. Res. Comm. 105, 717-723 (1982),

Iwanaga et al, International symposium on Pyrogen, 84-84 (Jun. 23-26,1987),

Aketagawa et al. J. Biol. Chem. 261, 7354-7365 (1986),

Hao, U.S. Pat. No. 4,677,194 (Jun. 30, 1987), and

Nachum et al, J. Inv. Path. 32, 51-58 (11978).

Tanaka et al, Iwanaga et al, and Aketagawa et al each conducted researchon an anti-LPS factor or endotoxin binding protein isolated from ahorseshoe crab system. Based on experimental work done in the inventors'laboratory, it appears that a protein involved in the present inventionis the same as that isolated by Iwanaga et al and Tanaka et al. However,these publications do not say anything about pharmaceutical utility ofthe endotoxin binding/neutralizing protein, and it is difficult topredict in vivo activity based on in vitro experimentation. In fact,neither of these references suggests a practical utility for theanti-LPS factor, and in view of the unpredictable nature of in vivoactivity, it has previously not been appreciated that the endotoxinbinding/neutralizing protein could be used in a pharmaceuticalcomposition. Furthermore, none of the references disclose the use of theendotoxin binding/neutralizing protein for an endotoxin assay, asdisclosed in the present invention. It is notable that the presentinventors have also discovered certain endotoxin binding/neutralizingprotein variants which have amino acid structures that are differentfrom the anti-LPS factor disclosed in the above-described publications.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to providepharmaceutical compositions containing therein an endotoxinbinding/neutralizing protein isolated from a horseshoe crab, whichcompositions are capable of binding and neutralizing endotoxin in vivo.

It is yet another object of this invention to provide pharmaceuticalcompositions capable of binding and neutralizing endotoxin in vivo andcontaining therein an endotoxin binding/neutralizing protein.

It is yet another object of the present invention to provide a methodfor reducing endotoxin concentration and/or neutralizing endotoxinactivity in vivo.

It is yet another object of the present invention to provide a methodfor purifying an endotoxin binding/neutralizing protein from a horseshoecrab.

It is yet another object of the present invention to provide a method ofassaying for endotoxin in a fluid.

These and other objects of the present invention which will hereinafterbecome more readily apparent, have been provided by purifying anendotoxin binding/neutralizing protein from the horseshoe crab Limuluspolyphemus, and discovering that it has the capability of binding to andneutralizing endotoxin in vivo. It has also been recognized that thispurified endotoxin binding/neutralizing protein is useful in an assayfor endotoxin. The present inventors have also discovered that there arecertain structural variants of the Limulus polyphemus endotoxinbinding/neutralizing protein, and it is expected that these materialswill also possess endotoxin binding/neutralizing ability such that theymay be used in the other aspects of the present invention as well.

BRIEF DESCRIPTION OF THE FIGURES

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 shows cation exchange chromatographic purification of Limulusendotoxin binding protein using CM-Sepharose resin. Peak 1: flowthrough, Peak 2: 0.25M NaCl elution, Peak 3: 1M NaCl elution.

FIG. 2 shows reversed phase chromatographic purification of Peak -1)from FIG. 1. Peak A: 25% isopropanol (IPA) elution, Peak B: 0.35% IPAelution, Peak C: 50% IPA elution, Peak D: column wash with 100% IPA.

FIG. 3 is a plot of apparent endotoxin concentration (measured by LAL)versus protein concentration. Such a plot can be used to assess proteinspecific endotoxin inactivating/neutralizing activity. Specific activityis expressed as the amount of protein needed to achieve 50% reduction ofendotoxin activity in the assay system (5 nanograms of endotoxin Per 100microliters). For convenience, units are expressed as microgramsprotein×10⁵ needed to reduce 5 nanograms activity/100 microliters ofstandard endotoxin by 50%.

FIG. 4 shows endotoxin removal endotoxin binding/neutralizing proteinimmobilized on a falten.

FIG. 5 shows fluorescence excitation and emission spectra of Limulusendotoxin binding/neutralizing protein.

FIG. 6 shows successive decreases in the emission spectra of Limulusendotoxin binding/neutralizing protein upon successive additions ofendotoxin.

FIG. 7 shows lack of-successive decreases in the emission spectra ofhuman serum albumin upon successive additions of endotoxin (negativecontrol).

FIG. 8 shows lack of successive decreases in the emission spectra ofpeak B, FIG. 2 upon successive additions of endotoxin (negativecontrol).

FIG. 9 shows a fluorescence titration of Limulus endotoxinbinding/neutralizing protein at pH 3.86. K_(D) =2.41 micromolar.

FIG. 10 shows fluorescence titration of Limulus endotoxinbinding/neutralizing protein at pH 6.91. K_(D) =0.51 micromolar.

FIG. 11 shows fluorescence titration of Limulus endotoxinbinding/neutralizing protein at pH 8.80. K_(D) =2.40 micromolar.

FIG. 12 shows the effect of pH on dissociation constant of Limulusendotoxin binding/neutralizing protein.

FIG. 13 is a comparison of immobilized Limulus endotoxinbinding/neutralizing protein compared to two immobilized polymyxinresins in proteinaceous and non-proteinaceous solutions.

FIG. 14 demonstration of activity of Limulus endotoxinbinding/neutralizing protein in the presence of total human serum. Thedashed line represents serum alone as a control (68 nanograms ofendotoxin per ml inactivated/neutralized). The solid line representsserum plus 25 micrograms/ml added endotoxin binding/neutralizing protein(1600, nanograms endotoxin per ml inactivated/neutralized).

FIG. 15 provides the amino acid sequence of the endotoxinbinding/neutralizing protein isolated from Limulus polyphemus inaccordance with the present invention (SEQ. ID. NO. 1).

FIG. 16 sets forth a DNA sequence (SEQ. ID. NO. 2) encoding a proteincorresponding to the endotoxin binding/neutralizing protein of thepresent invention having attached to its amino terminus the tetrapeptideGlu--Ala--Glu--Ala. The so modified endotoxin binding/neutralizingprotein is SEQ. ID. NO. 3. This DNA sequence is equipped with yeastpreferred codons and possesses unique restriction enzyme recognitionsites for convenient modification of the sequence.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The endotoxin binding/neutralizing protein of the present invention maybe isolated from any horseshoe crab. For example, any of the four knownspecies of horseshoe crabs could be used. These species are:

Limulus polyphemus

Tachypleus gigas

Tachypleus tridentatus

Carcinoscorpius rotundicauda

Especially preferred among these is Limulus polyphemus, the horseshoecrab which is found along the North American coast. The endotoxinbinding/neutralizing protein may be isolated by a procedure which issummarized hereinbelow. The procedure is illustrated for the Limulushorseshoe crab, but these procedures may be applied to any knownhorseshoe crab, as recited herein.

It was surprisingly found that the cellular debris produced duringlysate production from Limulus amebocytes contains significant amountsof the endotoxin binding/neutralizing protein. The cellular debris wasfound to have even more activity than lysate itself. By "cell debris" ismeant the insoluble material remaining when Limulus amebocytes are lysedby hypotonic shock. It includes nuclei, cell debris and some lysateproteins. The majority is insoluble material. Hypotonic shock may beaccomplished by treating the Limulus amebocytes with endotoxin-freedistilled water, preferably at 4° C., with shaking (e.g., for 12 h). Themixture is centrifuged, separating the lysate (soluble supernatant) fromthe cell debris.

The next step is to extract from the cell debris the Limulus amebocytebinding protein. Many solvents have been tested, including water,several alcohols (e.g., methanol, ethanol, isopropanol, and butanol),acetone, chloroform, acetonitrile, acids (e.g., HCl and H₂ SO₄), bases(e.g., NaOH and ethanolamine), salts (e.g., NaCl), and detergents (e.g.,Tween, triton. X-100, and SDS). The best results were obtained withdenaturants such as urea and guanidine hydrochloride. Surprisingly, sixmolar solutions of denaturants were effective in extracting the proteinwithout affecting biological activity. This concentration of denaturantwould be expected to inactivate most proteins. Thus, the first step inthe purification procedure herein is extraction of cellular debris fromLimulus amebocyte lysate with a denaturant to produce an extract. Theconcentration of the denaturant can range from 1N to 10M. A morepreferred range would be 3M to 8M. A most preferred concentration isaround 6 molar. Urea is the preferred denaturant, because it can be madefree of endotoxin by ultrafiltration and it is not readily contaminatedby endotoxin containing bacteria.

Preferably, prior to loading the extract onto a cation exchange column,an ultrafiltration step is performed. This step is also accomplishedusing urea or another denaturant as described above for extraction. Inparticular, the extract from the cell debris is crudely filtered with afilter aid such as diatomaceous earth (e.g., Celite®, Manville Corp.) orone of the cationic or anionic polymeric particles in colloidalsuspension (e.g., Biocryl®, Supelco division of Rohm and Haas Corp.),then passed through an ultrafiltration membrane. Ultrafiltrationmembranes may be used in any known form (e.g., plain film, hollow fiber,tubular and spiral) and may be made of any material. Preferred materialsare polysulfone, polyvinylidene fluoride, polyacrylonitrile, nylon, orcellulose. Most preferred is a polysulfone, flat or hollow fiber typeultrafiltration membrane. The preferred molecular weight cut-off of theultrafiltration membrane is 20,000 daltons to 50,000 daltons as can becommercially obtained from Millipore, Filtron or other membrane filtermanufacturers. The most preferred membrane has a 30,000 dalton cut-off.The Limulus endotoxin binding/neutralizing protein is now in thefiltrate at a very low concentration. The filtrate from the firstmembrane is concentrated using a second ultrafiltration membrane havinga molecular weight cut-off of 5,000 to 10,000 daltons, preferably an8,000 dalton cut-off membrane. The second ultrafiltration membrane maybe the same or different material as the first ultrafiltration membrane,and is preferably made of polysulfone. The principle of operation is thesame as the 30,000 cut-off membrane, however, the endotoxinbinding/neutralizing protein is now in the retentate with all otherproteins greater than e.g., 8,000 daltons.

After the above step, the retentate is subjected to cation exchangechromatography using cellulose, cross-linked agarose orhydrophilic-vinyl polymer resins derivatized with carboxymethyl (CM),Sulphopropyl (SP), Sulphonate (S), or other anionic group. These can beobtained from several manufacturers including Pharmacia (Sephadex®,Sepharose® breaded agarose), Tosoh Corp. (Toyopearl®) or BioRad(Cellex®, Bio-Gel). The most preferred resin is CM-Sepharose®. The pH ofloading and elution buffers can range from 4 to 8, with the mostpreferred range of pH 5 to 5.5. In this step also, urea (or anotherdenaturant as described above for extraction) is an importantconstituent. All column elution buffers must contain urea (at leastabout 3 molar) to elute the endotoxin binding protein cleanly from thecolumn.

The elution from the column is accomplished by a step gradient of a saltsuch as ammonium chloride, potassium chloride or sodium chloride. Sodiumchloride is preferred. FIG. 1 shows the results of the CM Sepharose®chromatographic step. The preferred concentration of urea in the eluentused in this step is 1M to 6M. A preferred concentration of urea in thisstep is 2M to 4M. A most preferred concentration is 2.5M to 3.5M. Whensodium chloride is used in the eluant, biological activity elutes at aconcentration of sodium chloride of from around 0.5 to 1 molar.

After the cation exchange step, the salt-eluted peak of activity isapplied to a reversed-phase column. The preferred method employs a resinhaving 4, 8, or 18 carbon chains (C-4, C-8 or C-18, respectively). Themost preferred method employs a C-4 resin commercially available fromsuch resin manufacturers as Vydac, Waters, Tosoh Corp., etc. The proteinis eluted by a step gradient of, for example, isopropanol andtrifluoroacetic acid (TFA). The concentration of trifluoroacetic acid isideally 0.2%, but can range from 0 to 0.4%, preferably 0.15 to 0.25%.FIG. 2 shows the location of the activity during treatment on thereversed-phase column. The reversed-phase column step effects desaltingand further purifies the material to virtual homogeneity. The Limulusprotein is remarkably stable to the organic solvents and low pH of thiscolumn. The protein is stable over a pH range of 1-3 in the presence ofTFA. The material may now be lyophilized. The purified Limulus proteinhas an isoelectric pH (pI), which is greater than 10, indicating a verybasic protein. The molecular weight of the protein as determined bySDS-PAGE is 12,500±100. The first three N-terminal amino acids of thispurified material are

    Asp--Gly--Ile

This protein is the preferred protein used in accordance with thepresent invention. Its amino acid seguence is set forth in FIG. 15 whereit is identified as SEQ. ID. NO. 1.

It should be noted that Watson and Sullivan, U.S. Pat. No. 4,107,077,teaches that there is an increase in sensitivity of the lysate byorganic extraction with chloroform. It appeared logical that a proteinwhich inactivated endotoxin would appear as an inhibitor in theendotoxin assay. Thus, it was hoped that the protein was the inhibitorwhich is extracted into chloroform and could be recovered from thatextract. However, surprisingly, this was not successful. Since theprotein is fairly hydrophobic, it may be in the organic extract, butdenatured in some way.

The majority of the purified material had an amino acid sequence whichappears to be identical to a protein from Limulus that was isolated byTanaka et al. The material was subsequently purified by Iwanaga et al,as reported in the International Symposium on Pyrogen, held Jun. 23-26,1987 in China. However, in the latter case, a different purificationprocedure was employed.

In addition to a protein having an apparently identical amino acidsequence to that reported in Iwanaga et al, some related proteins werealso purified, which have different amino acid sequences. These variantproteins have not previously been described. In a first protein, on theN-terminal thereof, a serine rather than an aspartic acid residue islocated. Furthermore, an asparagine is located in the second positionfrom the N-terminal rather than a glycine as reported in Iwanaga et al.Therefore, a protein having an initial amino acid sequence of

    Ser--Asn--Ile--Trp--Thr

is also part of the present invention. Other possible proteinderivatives are those beginning with

    Asp--Asn--

    Ser--Gly--, and

    Ser--Asn--,

and also a protein having an N-terminal asparagine with one less aminoacid than the natural sequence. Each of these variant proteins is alsopart of the present invention, and they may be used in each aspect ofthe invention described hereinbelow. They will be referred to asendotoxin binding/neutralizing protein variants (or variant proteins forshort), as distinguished from the natural sequence endotoxin bindingprotein, which is the protein having the amino acid sequence reported inIwanaga et al.

The variant proteins were not separated from each other. Rather, uponsequencing the most purified samples, some positions were uniformly oneamino acid, while other positions showed major variation. This isinterpreted as a mixture of proteins showing microheterogeneity. It isconsistent with the existence of a gene family for these proteinsindividual gene sequences are very homologous, but not identical. Itremains to be seen if their individual specific activities varysignificantly.

The present invention also encompasses DNA sequences which encode thepolypeptides of the present invention, vectors containing these DNAsequences, and microorganisms such as E. coli, yeast, etc., transformedwith the vectors. The transformed microorganisms can be used to producelarge quantities of the polypeptide materials, including the variantproteins described above. The genes of this invention may be altered soas to maximize codon expression in a given host.

The DNA sequences used in accordance with the present invention may beobtained in any manner known in the art, such as cloning and/or DNAsynthesis. Various methods for synthesizing both DNA and RNA sequencesare discussed in "Synthesis and Applications of DNA and RNA", edited byS. A. Narang, Academic Press Inc. (1987), which is hereby incorporatedby reference. Once obtained these gene sequences may be expressed inmicroorganisms using known methodology. See, e.g., Maniatis et al,"Molecular Cloning: A Laboratory Manual" Cold Spring Harbor Laboratory(1982), which discusses cloning and expression methodologies and whichis hereby incorporated by reference.

In accordance with the preferred embodiment of the present invention, aprotein encoded by SEQ. ID. NO: 3 is produced in a yeast host where itmay be produced as a glycoprotein comprising the amino acid sequence ofthe endotoxin binding protein of the present invention (SEQ. ID. NO. 1)to which the tetrapeptide "Glu--Ala--Glu--Ala" is covalently attached tothe amino terminal of the endotoxin binding protein. This glycoproteinencoded by SEQ. ID. NO: 2 is excreted by the yeast host.

Procedure for Assaying for Endotoxin Binding Activity

To follow the purification of the endotoxin binding/neutralizing proteinduring the above purification steps, assays can be performed insolution. One publication describing typical assays for LR₅₀ values isNovitsky et al, J. Clin. Microbiol. pp. 211-216 (1985). A specificprocedure follows:

A 96-well microtiter plate is used. Protein fractions are seriallydiluted across the top row and mixed with a standard endotoxin solution.There is another series of dilutions done on all samples to get withinrange of the assay, but basically the endotoxin recovered in the wellafter mixing with a suspected endotoxin inactivating protein ismeasured. This is compared to negative controls and an endotoxinstandard curve. It is found that the added endotoxin is inactivated atthe high protein concentrations. As the protein is diluted across theplate, a point is reached where it is too dilute to inactivate the addedendotoxin. This dilution is a measure of the protein specific activity.FIG. 3 represents a graphical form of such experiments. In FIG. 3, thedotted curve represents the crude extract after ultrafiltration, and thesolid line is the most highly purified fraction from the reversed-phasecolumn.

In Vivo Activity of the Endotoxin Binding/Neutralizing Protein of thePresent Invention

The endotoxin binding protein of the present invention (and variantsthereof) may be incubated with endotoxin, and then administered toanimals, and the bound endotoxin is not found to cause any pyrogenicityin vivo. The details of the endotoxin test are presented hereinbelow inthe examples section.

Surprisingly, and even less predictably, the present endotoxin bindingprotein may be administered to an animal after the animal has alreadybeen exposed to endotoxin, and the endotoxin binding protein can reversethe effects of free endotoxin in vivo. Moreover, the endotoxinbinding/neutralizing protein may be administered to an animal beforecontact with endotoxin by the animal, and the endotoxinbinding/neutralizing protein will exert a protective effect against theeffects of endotoxin. Accordingly, the endotoxin binding/neutralizingprotein of the present invention may be formulated into a pharmaceuticalcomposition for treating an animal in vivo, so as to exert a therapeuticeffect if endotoxin is present in the animal, or to exert a protectiveor preventive effect, if the animal should come into contact withendotoxin later. Details on the experimental tests showing the in vivoeffects of the endotoxin binding/neutralizing protein of the presentinvention are also presented in the examples section hereinbelow.

It should be noted that in vivo activity of this type would have beenunpredictable based on the bare disclosure of the in vitro endotoxinbinding/neutralizing capability of this protein. According to Nachum(described above), there were two possibilities for the removal ofendotoxin by lysate proteins, binding or enzymatic cleavage ofendotoxin. The inventors looked for degradation products and found none(specifically, free fatty acids that could have been released byesterases). Subsequent work has found binding to be the mechanism. It ismore expected that the endotoxin would be inactivated in vivo by anenzymatic action the molecule would be structurally different. However,by only binding, one would assume the entire endotoxin structure wouldstill be present and available to trigger the normal biological responseto the toxin. The noted inactivation in vitro might easily have been anartifact of an artificial assay system--perhaps a conformational changein the bound endotoxin not reacting with the LAL reagent, or it is alsopossible that the Limulus endotoxin binding protein was inhibiting theclotting reaction by itself (since it is present in lysate, thispossibility is hard to rule out without the in vivo results).

Although it might be possible for the endotoxin binding protein of thepresent invention to be administered in vivo without any additivesthereto, it is preferably mixed with a carrier, and if necessary, otheradjuvants.

By the term "carrier" as used herein is meant a synthetic or natural,inorganic or organic substance which is added to the endotoxin bindingprotein of the present invention to assist the active ingredient inreaching the location to be treated therewith and to facilitate storage,transportation and handling of the active ingredient.

Among suitable liquid carriers, there may be included aromatichydrocarbons such as benzene, toluene, xylene, cumene, etc., paraffinichydrocarbons such as mineral oil and the like, halogenated hydrocarbonssuch as carbon tetrachloride, chloroform, dichloroethane and the like,ketones such as acetone, methyl ethyl ketone, etc., ethers such asdioxane, tetrahydrofuran and the like, alcohols such as methanol,propanol, ethylene glycol and the like, dimethyl formamide,dimethylsulfoxide, water, etc. Mixtures of any number of liquid carriersare also envisioned. Upon dissolution of lyophilized active ingredientwith unbuffered pyrogen free distilled water or saline or phosphatebuffered saline (pH 6.5 to 7.5), the protein is not completely soluble.In order to avoid undesirable physiological side-effects which mightresult from such suspensions, the pH may be adjusted to slightlyalkaline pH (pH 8 to 9) at which the material becomes water clear. Forthis reason, the most preferred liquid carrier is pyrogen free distilledwater or saline adjusted to an alkaline pH.

In order to enhance the effectiveness of the compound according to thisinvention, it is possible to use such adjuvants as given below eithersingly or in combination in accordance with the purpose of eachapplication thereof while taking into consideration the form of theirpreparation and their field of application.

Namely, exemplary adjuvants may include anionic surfactants such asalkyl sulfates, aryl sulfonates, succinates, polyethylene glycol alkylaryl ether sulfates, and the like, cationic surfactants such asalkylamines, polyoxyethylene alkylamines, etc., non-ionic surfactantssuch as polyoxyethylene glycol ethers, polyoxyethylene glycol esters,polyol esters and the like, and amphoteric surfactants. Encapsulation ormicroencapsulation of the active ingredient in liposome vesicles is alsowithin the scope of this invention.

Examples of stabilizers, thickeners, lubricants and the like areisopropyl hydrogen-phosphate, calcium stearate, wax, casein, sodiumalginate, serum albumin, other blood proteins, methylcellulose,carboxymethylcellulose, gum arabic, etc. It should be kept in mind thatthese ingredients are not limited to the recited examples.

The active materials according to the present invention can beadministered by any route, for example, orally, parenterally,intravenously, intradermally, subcutaneously, or topically, in liquid orsolid form. Preferably, the route of administration is intravenously.

The solutions or suspensions may also include the following components:a sterile diluent such as water for injection, saline solution, oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple base vialsmade of glass or plastic.

While dosage values will vary with the specific severity of the diseasecondition to be alleviated, good results are achieved when the compoundsdescribed herein are administered to a subject requiring such treatmentas an effective oral, parenteral or intravenous dose of from 100 to10,000 units per nanogram of measured endotoxin per day per patient. Aparticularly preferred effective amount is about 1000 to 5000 units pernanogram of measured endotoxin per day per patient. Most preferred isthe administration of 0.1 to 100 mg of endotoxin binding protein/kg ofbody weight per day per patient. It is to be understood, however, thatfor any particular subject, specific dosage regimens should be adjustedto the individual need and the professional judgment of the personadministering or supervising the administration of the aforesaidcompound. It is to be further understood that the dosages set forthherein are exemplary only and they do not limit the scope or practice ofthe invention. The dosages may be administered at once, or may bedivided into a number of smaller doses to be administered at varyingintervals of time.

Specific diseases which could be treated include septicemia, toxicshock, and any other condition, especially gram-negative bacterialinfections, which is accompanied by an increase in in vivo endotoxincontent. Other examples are endotoxin-related arthritis, gonorrhea,periodontal disease, spinal meningitis, and infections of amnioticfluid.

Although this invention and its preferred embodiments are primarilyaddressed to use in humans, veterinary use is also encompassed by theinvention. In this regard, the active ingredient may be administered toreduce or prevent the pyrogenic or other ill effects of endotoxin invivo in, for example, dogs, cats, horses, cattle, sheep, and rabbits.Administration to laboratory animals, such as mice and rats, in order toprevent or reduce effects of endotoxin, is also contemplated.

Although polyclonal and monoclonal antibodies to endotoxin have alsobeen developed for this purpose and are the subject of ongoingdevelopment, the endotoxin binding protein of the present invention hasadvantages over antibodies which make it a preferred candidate for atherapeutic. The binding constant is very high and its low molecularweight may be less antigenic. In a recent paper, Greisman and Johnston,J. Infect. Disease 157:54-64 (1988), it has been shown that at least oneanti-serum against endotoxin is ineffective in reducing the effects ofendotoxin in vivo. In contrast, the in vivo experiments in the presentinvention have shown that the endotoxin binding protein may inactivateendotoxin in both rabbit and human serum.

Removal of Endotoxin From Solutions

Endotoxin contamination of pharmaceuticals is a problem toward whichmany removal protocols have been directed. Many of these are summarizedin Biopharm, April, 1988, pages 22-29, and the references cited therein.They all fall short when the contaminated material is or contains a highmolecular weight substance, such as protein. Most of the existingtechnology for endotoxin removal (ultrafiltration, ion exchange,affinity chromatography) does not separate protein and endotoxin. Thepresent invention involves using the endotoxin binding protein (e.g.,from Limulus) as an immobilized affinity ligand to achieve thisseparation. The preferred solid support for covalently coupling theendotoxin binding protein is cellulose, agarose or other hydrophilicpolymer derivatized to contain carboxyl, hydroxyl, amino, epoxide orother chemically reactive group to which proteins may be covalentlyattached using well known coupling chemistry. These methods includeusing water soluble carbodiimide or carbonyldiimidazole, glutaraldehyde,etc. The most preferred support is the hydrophilic vinyl polymer inpellicular or membrane form containing carboxy-methyl groups activatedwith water-soluble carbodiimide. The inventors have attached the bindingprotein covalently to chromatographic beads and the protein retains itsbiological activity. Furthermore, by mixing the beads with endotoxincontaminated proteins, the solution could be detoxified. This was doneby mixing the beads in batch mode in test tubes, or by packing a columnwith the beads and passing the solutions over it.

The protein could also be covalently attached to filter membranes forthe same purpose. FIG. 4 shows an experiment with such a filter. A 40 mlsolution containing approximately 130 pg/ml endotoxin was recirculatedthrough a filter containing the binding protein. There was an initiallarge drop in the toxin concentration that was further reduced as thematerial recirculated.

The present inventors further conducted a direct comparison of twoimmobilized polymyxin resins compared to the present ligand on similargel particles. The experiment took 100 microliter gel volumes of each ina microcentrifuge tube and added 100 nanograms of E. coli endotoxin in 1ml of various solutions to each. The tubes were mixed end over end forone hour. In 10 mM Tris, all three performed equally well. In 25% humanserum albumin (HSA) and 5% HSA, the present endotoxin binding proteinwas able to remove significantly more endotoxin. Interestingly, indistilled water, polymyxin was superior. Thus, the present endotoxinbinding protein exhibits surprisingly superior performance inprotein-containing solutions. Other possible proteins contaminated byendotoxin are HSA, interferon, interleukins, growth factors, hormones,proteases, TPA, TNF, monoclonals, EGF, insulin, and erythropoietin.

Extracorporeal Treatment of Septic Shock

Because of the ability of the endotoxin protein to function in proteinsolutions and human serum, an affinity membrane or hollow fiber may beconstructed for the purpose of removing endotoxin from the blood ofpatients extracorporeally. In such a mode, similar to kidney dialysis,blood containing elevated concentrations of endotoxin from a variety ofpotential clinical conditions is circulated through an affinity devicesuch that the serum is brought into direct contact with endotoxinbinding protein covalently bound to the membrane. Since the amount ofendotoxin binding protein being released into the general circulation ismuch reduced from an intravenous application, potential side effects areminimized.

Assay for Binding Affinity and Application of the EndotoxinBinding/Neutralizing Protein in an Alternative Endotoxin Assay

A known optical property of all proteins is the ability of some aromaticamino acids to fluoresce when excited at certain wavelengths. Thepresent inventors looked specifically at tryptophan residues. These areexcited at around 283 nm and fluoresce at around 350 nm. See FIG. 5.

The inventors examined a fluorescence maxima at 350 nm of the endotoxinbinding protein at various concentrations of endotoxin. There was aproportional quenching of that fluorescence, indicative of a tightassociation. See FIG. 6. Other proteins as negative controls showed nosuch proportional change. See FIGS. 7 and 8. From this data, theinventors have been able to calculate the binding affinity constantsbetween the present protein and endotoxin. See FIGS. 9-11.

The fluorescence quenching experiment may serve as the basis for anassay for endotoxin based on the physical change of a single proteinupon exposure to endotoxin. The inventors have found that the quenchingeffect observed upon addition of endotoxin to the endotoxin bindingprotein does not appear to experience saturation up to a highconcentration level of added endotoxin. In other words, although onewould expect the relationship between concentration of endotoxin andquenching to be a nonlinear one, the linearity of the relationshipextends over a remarkably wide range of concentration of endotoxin. Thisunexpected aspect of the present assay enables the assay to be used overa wider range of endotoxin concentrations than could have been expected.Ranges of the ratio of endotoxin binding protein to endotoxin that areexpected to be effective are 0.1:1 to 5000:1, preferably 1:1 to 2000:1,most preferably 10:1 to 1000:1.

In a preferred embodiment of the present endotoxin assay using thepresent endotoxin binding protein, the response is amplified using abiosensor. Biosensors rely on a specific binding ligand immobilized on asolid (e.g., quartz) chip. Electrical potential between two electrodes,or the acoustic wave perturbation between two electrodes is measured.

The current method for measuring endotoxin is the Limulus amebocytelysate assay. This involves a cascade of enzymes which are activated byendotoxin and result in the formation of a gel, analogous to a mammalianblood clot. Since multiple enzymes are involved, many substancesinterfere with the overall reaction, causing enhancement or inhibition.In addition, the key enzymes of the cascade are proteases so otherproteolytic enzymes, such as trypsin, can cause gel formation. Toeliminate these problems, samples containing interfering substances mustbe diluted beyond the point where they interfere. In many cases thedilution factor can be hundreds or thousands. The net effect is todecrease the sensitivity in measuring absolute endotoxin concentrationsin those samples.

One of the promising potentials that a single endotoxin binding proteinhas is to eliminate the multiple interfering effects seen in the LALassay. Even if the sensitivity were one tenth that of TAL,, the lack ofa need to dilute would compensate, making the sensitivity equivalent.Biosensors take advantage of specific binding affinities of suchmolecules. Antibodies are an example which have been used sound to thesurface of a silicon chip, they bind to their immunogen and cause achange in the surface which is measured electrically. Depending on thetype of sensor, capacitance, resistance or acoustic wave changes aremeasurable. Depending on the concentrations and volumes of the samples,the time required for each measurement can be from seconds to minutes.

The electronic component of the biosensor could measure voltage(potentiometric), current (amperometric), light, sound, temperature ormass (piezoelectric). The biosensors of the present invention can bebased on technology which is known, such as described in Biotechnology5, 437-440 (1987), and Transducers 1987, the abstract entitled"Development of a Surface Acoustic Wave Biosensor", and "Recent Progressin Biosensor Development", Hall, E. A. H., Int. J. Biochem. 1988, 20(4),357-62, each of which is incorporated herein by reference. Biosensorinformation is also contained in U.S. Pat. Nos. 4,721,677, 4,680,268, H0,000,201, and 4,614,714, each of which is incorporated herein byreference.

An actual device employing a biosensor would be similar to a flow cell.The sample would enter, be exposed to the reactive surface and ameasurement made. Depending on the chemistry of binding, the boundligand could be washed off to regenerate the surface or left on tomeasure a cumulative response. This may be adapted to an in-line deviceto monitor endotoxin contaminating events.

Due to the compact nature of the electronics, a portable field unit isfeasible. This could be used on-site for checking water purity orprocess cleanliness during pharmaceutical manufacture, kidney dialysisunit pyrogen checks, or any other application where the conventional LALis used now. Since endotoxin is a component of gram negative bacterialcell walls, binding of whole bacteria should be measurable. An extensionof this technology should make possible remote sensors to monitor waterquality in the environment.

By coupling the binding protein to latex microspheres using a chemistrysimilar to that used to immobilize the protein on chromatographic resinsor membranes, another method to quantitate endotoxin is constituted. Byexposing latex microspheres coated with endotoxin binding protein (e.g.,from Limulus) to endotoxin, the microspheres agglutinate. Suchagglutination is commonly employed in assays based on antibodies to thespecific ligand to be measured (Hechemy, K. E.; Michaelson, E. E.Laboratory Menacement 1984, 22(6):27,ff (Part I) and 22(7):26,f (PartII); Babson, A. L., Opper, C. A. and Crane L. J. American Journal ofClinical Pathology (1982), 77(4):424-9). In such techniques, theagglutination phenomenon is thought to be due to crosslinking betweenmultiple binding sites on the antibody molecule complexing with multiplesites on the antigen. Due to the high ratio of endotoxin binding proteinto endotoxin needed to be effective in our other solution experiments,it is thought the number of endotoxin binding sites on the protein isone or less. By covalently coupling many molecules of binding proteinper microsphere, a multiple binding site reagent is created which is nowpossible to function as a specific agglutinin. Alternatively, thebinding protein can be linked to one suspension of beads and endotoxinor lipid A linked to a second suspension. On mixing, these twosuspensions will agglutinate. This may also be employed as an assay forendotoxin in solution by the ability of the free endotoxin to inhibitsuch agglutination. In this case, agglutination is inverselyproportional to the unknown endotoxin concentration.

The invention now being generally described, it will now be illustratedin greater detail by the following examples, which are presented hereinfor illustration only and are not intended to be limiting of the presentinvention, unless so indicated.

EXAMPLES Example 1

Assay in of in vitro Inactivated Endotoxin by Pyrogen Testing inRabbits:

E. coli endotoxin was prepared at 100 nanograms per ml. in PhosphateBuffered Saline (PBS). This was mixed with 200 micrograms of LimulusEndotoxin Binding Protein and incubated at 37 degrees Celsius for onehour. Control solutions were prepared of endotoxin (100 nanograms/ml)only and PBS only. Three vials of each of the three solutions wereprepared containing 2 ml/vial. 1.5 ml of each solution was injectedintravenously into individual 3 kilogram rabbits and their bodytemperatures measured continuously for six hours with data pointsrecorded every 10 minutes. The final dose of endotoxin or inactivatedendotoxin was 50 nanograms/kilogram.

In rabbits, a 5 nanograms/kilogram dose of endotoxin elicits ameasurable fever response, showing a peak at one hour after injection.The present results showed the rabbits receiving only endotoxin at 50nanograms/kilogram did indeed develop a body temperature elevated 1.64(S.D. 0.236) degrees Celsius. Those rabbits receiving only PBS orendotoxin inactivated with Limulus Endotoxin Binding Protein maintaineda normal body temperature.

Example 2

Assay of in vivo Prophylactic Efficacy of Limulus Endotoxin BindingProtein:

Limulus Endotoxin Binding Protein is injected intravenously into rabbitsat two dose levels, 5 micrograms/kilogram and 50 micrograms/kilogram.Control animals receive injections of PBS. Fifteen minutes later, allanimals receive intravenous injections of 50 nanograms/kilogram E. coliendotoxin. Body temperature is monitored continuously over six hours andrecorded every 10 minutes. The normal course of increase in temperaturepeaking at one hour after injection is seen in control animalspreinjected with PBS only. Those animals receiving preinjection ofendotoxin binding protein at 50 micrograms/kilogram showed an increaseof 1.55 (S.D. 0.225) degrees Celsius. Animals receiving 5micrograms/kilogram demonstrated temperature increases of 1.85 (S.D.0.270).

Example 3

Assay of in vivo Therapeutic Efficacy of Limulus Endotoxin BindingProtein:

E. coli endotoxin is injected intravenously into 9 rabbits at a dose of50 nanograms/kg. After 15 minutes, three were injected intravenouslywith 5 micrograms Limulus Endotoxin Binding Protein, three were injectedintravenously with 50 micrograms Limulus Endotoxin Binding Protein andthree received PBS. Volumes of all injections were 0.5 ml/kg. Bodytemperatures are monitored for 6 hours and data collected every 10minutes. Animals receiving endotoxin and the PBS only, manifest thenormal peak fever response one hour after toxin administration. Thoseanimals receiving therapeutic injections of the Limulus protein exhibita much reduced fever response proportional to the amount of proteinwhich is administered.

Example 4

Endotoxin Inactivating Potential of Limulus Endotoxin Binding Protein inHuman Serum:

In order to assay the potential effectiveness of the endotoxin bindingprotein as a therapeutic or prophylactic agent for septic shock orrelated disorders in humans, the protein was tested for its ability toinactivate endotoxin in the presence of whole human serum. The assay wasconducted in a 96-well tissue culture multiwell elates as described indetail in Novitsky et al., J. Clin. Micro., 20: 211-216 (1985). To eachwell were added in order 0.05 ml of serum only or serum with 25micrograms/ml Limulus Endotoxin Binding Protein, 0.05 ml E. coliendotoxin solution. The plates were covered with Parafilm to preventevaporation, agitated on a mechanical vibrating platform for 15 secondsand incubated at 37 degrees Celsius. Multiwell plates were thenuncovered, and 0.1 ml of LAL was added to each well. The plates weresubsequently handled and read as described for the LAL endotoxin assay.Serum with the added endotoxin binding protein was able to inactivateincreasingly larger amounts of endotoxin (2,000 nanograms/ml) whileserum alone was able to bind and/or inactivate only 80 nanograms/ml.

Example 5

Universality of Endotoxin Type Inactivated by the Limulus EndotoxinBinding Protein.

In order to investigate the mechanism of the endotoxin bindingphenomenon and determine the breadth of endotoxin types against whichthe binding protein is effective, endotoxin from several species of Gramnegative bacteria as well as lipid A were tested. These includedendotoxin from Klebsielia pneumonias, Serratia marcescens, Salmonellaenteritidis, Escherichia coli 0113 wild type, E. coli rough mutant(J-5), Salmonella abortus equi, and Lipid A from S. minnesota Re 595.

The endotoxin binding protein was mixed with the endotoxins or lipid Aat a ratio of 5:1 to 1,000:1 in the presence of 10 millimolar Trisbuffer, pH 6-8. In all cases the measurable endotoxin activity aftermixing was reduced 85% to 99.5%.

Example 6

Summary:

Limulus Endotoxin Binding Protein (EBP) protects rats from the lethaleffects of lipopolysaccharide (LPS). The effects of premixed-LPS andEBP(1:1 wt/wt) were compared to those of EBP or LPS alone. The materialwas administered to rats in groups. The endotoxin group exhibited 30%mortality; the group which received the combined EBP+LPS exhibited nodeaths. In an in vitro experiment, vascular tissue was examined forcontraction defects after preincubation with endotoxin, protein, or amixture of endotoxin and protein. Endotoxin-incubated tissue exhibitedreduced contraction to norepinephrine; coincubation with limulus proteinprotected against the supression.

Hematocytes from Limulus polyphemus contain a 12,000 dalton amphipathicprotein with a high affinity for the lipid A portion of endotoxin. Thisprotein is part of an anti-infection pathway of aggregation where rapiddegranulation and clot formation are initiated when hematocytes areexposed to Gram-negative endotoxins. This endotoxin binding protein hasbeen isolated and sequenced (SEQ. ID. NO. 1). In vitro it binds toendotoxin with high affinity and neutralizes the lipopolysaccharide thuspreventing it from being detected in the Limulus amebocyte assay. In arecent study which shows that this protein can protect endothelial cellsin vitro from the toxic affect of endotoxin, the ability of the Limulusendotoxin binding-protein to protect rats from the toxic effects ofendotoxin was examined. Also, the ability of this protein to protectvascular tissue from the effects of endotoxin was examined in vitroexperiments. The results of both in vivo and in vitro experimentsdemonstrate that this protein exhibits a protective effect byneutralizing the endotoxin.

Methods:

Protection From Endotoxemia

Male Sprague-Dawley rats (230-450 g) were given light Halothaneanesthesia and then injected with the appropriate experimental treatmentvia the dorsal vein of the penis.

Groups of rats received one of the following treatments;

1) LPS (E. coli O111:B4, Sigma Chemical Company) in buffer, 0.15M NaClbuffered to pH 7.4 with 0.02M sodium phosphate,

2) A suspension of anti-LPS factor and LPS mixed 1:1 (wt/wt) in buffer,

3) Albumin mixed with LPS 1:1 (wt/wt), or

4) Only the Limulus protein incubated in buffer.

All solutions were incubated at 37° C. for one hour prior to injection.The volume injected (1.7 to 2.9 ml) depended on the weight of the ratand was adjusted to deliver an exact dose on a mg LPS/kg body weightbasis. Animals were maintained on standard rat chow and water adlibitum. Survival was followed to 24 hours.

Aortic Ring Contraction

Rats were sacrificed by decapitation and the thoracic aorta excised andsectioned into rings for measures of contractile performance. Tissuesfrom normal rats were incubated at 37° C. for 16 hours in DMEM(Dulbecco's Minimal Essential Medium) containing 5% fetal calf serum,100 U penicillin, 100 ug streptomycin, and gassed with 95% O₂ -5% CO₂.Experiments consisted of additions of either;

1) 10 ng/ml endotoxin,

2) 50 ng/ml endotoxin binding-protein,

3) a mixture of endotoxin and binding-protein (10 and 50 ng/ml), or

4) medium only during the 16 hr incubation period.

Contractile performance of rings suspended between two hooks in a 10 mlbath was assessed by measuring the tension as a function of cumulativedoses of norepinephrine (NE) covering a range of 1 nM to 30 uM.

    ______________________________________                                        Survival as a Function of Endotoxin/Neutralizing                              Protein (EBP)                                                                  ##STR1##                                                                     ______________________________________                                        EXP.sup.1 # 1                                                                  ##STR2##                                                                      ##STR3##                                                                     ______________________________________                                        LPS = (15 mg/kg, # 37F-4089) this lot # exhibits reduced toxicity             .sup.a p < .05 different from LPS Buffer                                      .sup.b p < .05 different from LPS albumin                                      ##STR4##                                                                     ______________________________________                                        EXP.sup.2 # 2                                                                  ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                     ______________________________________                                         ##STR8##                                                                     .sup.c,d no significant different but trend consistent (p < .1)               Survival as a Function of Native Endotoxin/Neutralizing                       Protein (nENP) and Recombinant Endotoxin                                      Binding/Neutralizing Protein (rENP)                                            ##STR9##                                                                     ______________________________________                                         ##STR10##                                                                     ##STR11##                                                                    ______________________________________                                         ##STR12##                                                                    .sup.d p < .05 different from LPS in buffer                                   .sup.e p < .05 different from LPS in albumin                                  *****                                                                     

The invention now being fully described, it will be appreciated that theinvention may be practiced otherwise than as specifically set forthherein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 101 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AspGlyIleTrpThrGlnLeuIlePheThrLeuValAsnAsnLeuAla                              151015                                                                        ThrLeuTrpGlnSerGlyAspPheGlnPheLeuAspHisGluCysHis                              202530                                                                        TyrArgIleLysProThrPheArgArgLeuLysTrpLysTyrLysGly                              354045                                                                        LysPheTrpCysProSerTrpThrSerIleThrGlyArgAlaThrLys                              505560                                                                        SerSerArgSerGlyAlaValGluHisSerValArgAsnPheValGly                              65707580                                                                      GlnAlaGlySerSerGlyLeuIleThrGlnArgGlnAlaGluGlnPhe                              859095                                                                        IleSerGlnTyrAsn                                                               100                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 331 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GAGGCTGAAGCTGACGGTATCTGGACCCAATTGATTTTCACTTTGGTT48                            GluAlaGluAlaAspGlyIleTrpThrGlnLeuIlePheThrLeuVal                              151015                                                                        AACATTTTGGCCACCTTATGGCAGTCCGGTGATTTTCAATTCTTGGAC96                            AsnIleLeuAlaThrLeuTrpGlnSerGlyAspPheGlnPheLeuAsp                              202530                                                                        CACGAATGTCACTACAGAATCAAGCCAACTTTCAGAAGATTGAAGTGG144                           HisGluCysHisTyrArgIleLysProThrPheArgArgLeuLysTrp                              354045                                                                        AAATATAAGGGTAAATTTTGGTGTCCATCTTGGACCTCTATTACTGGT192                           LysTyrLysGlyLysPheTrpCysProSerTrpThrSerIleThrGly                              505560                                                                        AGAGCTACCAAGTCTTCTAGATCCGGTGCTGTCGAACACTCTGTTAGA240                           ArgAlaThrLysSerSerArgSerGlyAlaValGluHisSerValArg                              65707580                                                                      AACTTCGTCGGTCCAGCTAAGTCTTCCGGTTTGATCACTGAAAGACAA288                           AsnPheValGlyProAlaLysSerSerGlyLeuIleThrGluArgGln                              859095                                                                        GCTGAACAATTCATTTCTCAATACAACTGATAAGCTTGAATTC331                                AlaGluGlnPheIleSerGlnTyrAsn                                                   100105                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 105 amino acids                                                   (B) TYPE: amino acid                                                          (C) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GluAlaGluAlaAspGlyIleTrpThrGlnLeuIlePheThrLeuVal                              151015                                                                        AsnIleLeuAlaThrLeuTrpGlnSerGlyAspPheGlnPheLeuAsp                              202530                                                                        HisGluCysHisTyrArgIleLysProThrPheArgArgLeuLysTrp                              354045                                                                        LysTyrLysGlyLysPheTrpCysProSerTrpThrSerIleThrGly                              505560                                                                        ArgAlaThrLysSerSerArgSerGlyAlaValGluHisSerValArg                              65707580                                                                      AsnPheValGlyProAlaLysSerSerGlyLeuIleThrGluArgGln                              859095                                                                        AlaGluGlnPheIleSerGlnTyrAsn                                                   100105                                                                        __________________________________________________________________________

What is new and desired to be secured by Letters Patent of the UnitedStates is:
 1. A method for reducing endotoxin contamination of amaterial suspected of containing endotoxin, comprising contacting saidmaterial with the endotoxin binding molecule having SEQ ID NO: 3 to forma complex between endotoxin and the endotoxin binding molecule, andseparating said complex from said sample.
 2. A method for theextracorporeal removal of endotoxin from blood, comprising contactingthe blood with the endotoxin binding protein having SEQ ID NO:3 to forma complex between the endotoxin and the endotoxin binding molecule, andseparating said complex from said blood.
 3. The method of claim 1 or 2,wherein said endotoxin binding protein is immobilized on achromatographic resin or on a membrane.
 4. The method of claim 1 or 2,wherein said endotoxin binding molecule is immobilized on latexmicrospheres.